Degradation pathway of bisphenol A: Does ipso substitution apply to phenols containing a quaternary alpha-carbon structure in the para position?

The degradation of bisphenol A and nonylphenol involves the unusual rearrangement of stable carboncarbon bonds. Some nonylphenol isomers and bisphenol A possess a quaternary alpha-carbon atom as a common structural feature. The degradation of nonylphenol in Sphingomonas sp. strain TTNP3 occurs via a type II ipso substitution with the presence of a quaternary alpha-carbon as a prerequisite. We report here a new degradation pathway of bisphenol A. Consequent to the hydroxylation at position C-4, according to a type 11 ipso substitution mechanism, the C-C bond between the phenolic moiety and the isopropyl group of bisphenol A is broken. Besides the formation of hydroquinone and 4-(2-hydroxypropan-2-yl) phenol as the main metabolites, further compounds resulting from molecular rearrangements consistent with a carbocationic intermediate were identified. Assays with resting cells or cell extracts of Sphingomonas sp. strain TTNP3 under an 18 02 atmosphere were performed. One atom of 180, was present in hydroquinone, resulting from the monooxygenation of bisphenol A and nonylphenol. The monooxygenase activity was dependent on both NADPH and flavin adenine dinucleotide. Various cytochrome P450 inhibitors had identical inhibition effects on the conversion of both xenobiotics. Using a mutant of Sphingomonas sp. strain TTNP3, which is defective for growth on nonylphenol, we demonstrated that the reaction is catalyzed by the same enzymatic system. In conclusion, the degradation of bisphenol A and nonylphenol is initiated by the same monooxygenase, which may also lead to ipso substitution in other xenobiotics containing phenol with a quaternary a-carbon.

Liquid ultraviolet matrix-assisted laser desorption/ionization - mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet -- matrix-assisted laser desorption/ionisation -- mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. The low-femtomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydroxybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and low-mass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.

Liquid ultraviolet matrix-assisted laser desorption/ionization mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet - matrix-assisted laser desorption/ ionisation - mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. U. Am. Soc. Mass Spectrom. 1998, 9, 166-174). The low-ferntomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydrox-ybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and lowmass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.

CPD records for revalidation: assessing fitness-to-practise using ‘revalidation standards’ and an Outcomes Framework

Background: Currently, all pharmacists and technicians registered with the Royal Pharmaceutical Society of Great Britain must complete a minimum of nine Continuing Professional Development (CPD) record (entries) each year. From September 2010 a new regulatory body, the General Pharmaceutical Council, will oversee the regulation (including revalidation) of all pharmacy registrants in Great Britain. CPD may provide part of the supporting evidence that a practitioner submits to the regulator as part of the revalidation process. Gaps in knowledge necessitated further research to examine the usefulness of CPD in a pharmacy revalidation Project aims: The overall aims of this project were to summarise pharmacy professionals’ past involvement in CPD, examine the usability of current CPD entries for the purpose of revalidation, and to examine the impact of ‘revalidation standards’ and a bespoke Outcomes Framework on the conduct and construction of CPD entries for future revalidation of pharmacy professionals. We completed a comprehensive review of the literature, devised, validated and tested the impact of a new CPD Outcomes Framework and related training material in an empirical investigation involving volunteer pharmacy professionals and also spoke with our participants to bring meaning and understanding to the process of CPD conduct and recording and to gain feedback on the study itself. Key findings: The comprehensive literature review identified perceived barriers to CPD and resulted in recommendations that could potentially rectify pharmacy professionals’ perceptions and facilitate participation in CPD. The CPD Outcomes Framework can be used to score CPD entries Compared to a control (CPD and ‘revalidation standards’ only), we found that training participants to apply the CPD Outcomes Framework resulted in entries that scored significantly higher in the context of a quantitative method of CPD assessment. Feedback from participants who had received the CPD Outcomes Framework was positive and a number of useful suggestions were made about improvements to the Framework and related training. Entries scored higher because participants had consciously applied concepts linked to the CPD Outcomes Framework whereas entries scored low where participants had been unable to apply the concepts of the Framework for a variety of reasons including limitations posed by the ‘Plan & Record’ template. Feedback about the nature of the ‘revalidation standards’ and their application to CPD was not positive and participants had not in the main sought to apply the standards to their CPD entries – but those in the intervention group were more likely to have referred to the revalidation standards for their CPD. As assessors, we too found the process of selecting and assigning ‘revalidation standards’ to individual CPD entries burdensome and somewhat unspecific. We believe that addressing the perceived barriers and drawing on the facilitators will help deal with the apparent lack of engagement with the revalidation standards and have been able to make a set of relevant recommendations. We devised a model to explain and tell the story of CPD behaviour. Based on the concepts of purpose, action and results, the model centres on explaining two types of CPD behaviour, one following the traditional CE pathway and the other a more genuine CPD pathway. Entries which scored higher when we applied the CPD Outcomes Framework were more likely to follow the CPD pathway in the model above. Significant to our finding is that while participants following both models of practice took part in this study, the CPD Outcomes Framework was able to change people’s CPD behaviour to make it more inline with the CPD pathway. The CPD Outcomes Framework in defining the CPD criteria, the training pack in teaching the basis and use of the Framework and the process of assessment in using the CPD Outcomes Framework, would have interacted to improve participants’ CPD through a collective process. Participants were keen to receive a curriculum against which certainly CE-type activities could be conducted and another important observation relates to whether CE has any role to play in pharmacy professionals’ revalidation. We would recommend that the CPD Outcomes Framework is used in the revalidation of pharmacy professionals in the future provided the requirement to submit 9 CPD entries per annum is re-examined and expressed more clearly in relation to what specifically participants are being asked to submit – i.e. the ratio of CE to CPD entries. We can foresee a benefit in setting more regular intervals which would act as deadlines for CPD submission in the future. On the whole, there is value in using CPD for the purpose of pharmacy professionals’ revalidation in the future.

Global transcriptome analysis and identification of differentially expressed genes after infection of Fragaria vesca with powdery mildew (Podosphaera aphanis)

Background: Podosphaera aphanis, the causal agent of strawberry powdery mildew causes significant economic loss worldwide. Methods: We used the diploid strawberry species Fragaria vesca as a model to study plant pathogen interactions. RNA-seq was employed to generate a transcriptome dataset from two accessions, F. vesca ssp. vesca Hawaii 4 (HW) and F. vesca f. semperflorens Yellow Wonder 5AF7 (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection. Results: Of the total reads identified about 999 million (92%) mapped to the F. vesca genome. These transcripts were derived from a total of 23,470 and 23,464 genes in HW and YW, respectively from the three time points (control, 1 and 8 DAI). Analysis identified 1,567, 1,846 and 1,145 up-regulated genes between control and 1 DAI, control and 8 DAI, and 1 and 8 DAI, respectively in HW. Similarly, 1,336, 1,619 and 968 genes were up-regulated in YW. Also 646, 1,098 and 624 down-regulated genes were identified in HW, while 571, 754 and 627 genes were down-regulated in YW between all three time points, respectively. Conclusion: Investigation of differentially expressed genes (log2 fold changes �5) between control and 1 DAI in both HW and YW identified a large number of genes related to secondary metabolism, signal transduction; transcriptional regulation and disease resistance were highly expressed. These included flavonoid 3´-monooxygenase, peroxidase 15, glucan endo-1,3-β-glucosidase 2, receptor-like kinases, transcription factors, germin-like proteins, F-box proteins, NB-ARC and NBS-LRR proteins. This is the first application of RNA-seq to any pathogen interaction in strawberry

Interactions between the powdery mildew effector BEC1054 and barley proteins identify candidate host targets

There are over 500 candidate secreted effector proteins (CSEPs) or Blumeria effector candidates (BECs) specific to the barley powdery mildew pathogen Blumeria graminis f.sp. hordei. The CSEP/BEC proteins are expressed and predicted to be secreted by biotrophic feeding structures called haustoria. Eight BECs are required for the formation of functional haustoria. These include the RNase-like effector BEC1054 (synonym CSEP0064). In order to identify host proteins targeted by BEC1054, recombinant BEC1054 was expressed in E. coli, solubilized, and used in pull-down assays from barley protein extracts. Many putative interactors were identified by LC-MS/MS after subtraction of unspecific binders in negative controls. Therefore, a directed yeast-2-hybrid assay, developed to measure the effectiveness of the interactions in yeast, was used to validate putative interactors. We conclude that BEC1054 may target several host proteins, including a glutathione-S-transferase, a malate dehydrogenase, and a pathogen-related-5 protein isoform, indicating a possible role for BEC1054 in compromising well-known key players of defense and response to pathogens. In addition, BEC1054 interacts with an elongation factor 1 gamma. This study already suggests that BEC1054 plays a central role in barley powdery mildew virulence by acting at several levels.